Complex Formation between S100B Protein and the p90 Ribosomal ...

Complex Formation between S100B Protein and the p90Ribosomal S6 Kinase (RSK) in Malignant Melanoma IsCalcium-dependent and Inhibits Extracellular Signal-regulated Kinase (ERK)-mediated Phosphorylation of RSK*

Received for publication, February 27, 2014, and in revised form, March 11, 2014 Published, JBC Papers in Press, March 13, 2014, DOI 10.1074/jbc.M114.561613

Kira G. Hartman‡1, Michele I. Vitolo§¶1, Adam D. Pierce�**, Jennifer M. Fox‡ ‡‡, Paul Shapiro¶§§, Stuart S. Martin‡§¶,Paul T. Wilder§¶�2, and David J. Weber‡¶�**3

From the �Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland21201, the ‡Program in Molecular Medicine, University of Maryland Graduate Program in Life Sciences, Baltimore, Maryland21201, the §Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, the **Center forBiomolecular Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201, the §§Department ofPharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, the ¶University of MarylandMarlene and Stewart Greenebaum NCI Cancer Center, Baltimore, Maryland 21201, and the ‡‡Center for Stem Cell Biology andRegenerative Medicine, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland 21201

Background: S100B is overexpressed in malignant melanoma and contributes to cancer progression.Results: The S100B-RSK complex was found to be Ca2�-dependent, block phosphorylation of RSK at Thr-573, and sequesterRSK to the cytosol.Conclusion: The Ca2�-dependent S100B-RSK complex provides a new link between the MAPK and Ca2� signaling pathways.Significance: S100B inhibitors may restore normal MAPK and Ca2� signaling in malignant melanoma.

S100B is a prognostic marker for malignant melanoma.Increasing S100B levels are predictive of advancing diseasestage, increased recurrence, and low overall survival in malig-nant melanoma patients. Using S100B overexpression andshRNAS100B knockdown studies in melanoma cell lines, elevatedS100B was found to enhance cell viability and modulate MAPKsignaling by binding directly to the p90 ribosomal S6 kinase(RSK). S100B-RSK complex formation was shown to be Ca2�-dependent and to block ERK-dependent phosphorylation ofRSK, at Thr-573, in its C-terminal kinase domain. Additionally,the overexpression of S100B sequesters RSK into the cytosol andprevents it from acting on nuclear targets. Thus, elevated S100Bcontributes to abnormal ERK/RSK signaling and increased cellsurvival in malignant melanoma.

The S100 protein family consists of more than 20 membersexpressed in a tissue- and cell type-specific manner. One of theearliest discovered S100 family members, S100B, is a 21.5-kDasymmetric noncovalant homodimer found in melanocytes, glialcells, chondrocytes, and adipocytes (1, 2), with higher than nor-mal levels observed in malignant melanoma (3) and in severalother cancers (4, 5). Clinically, S100B is a prognostic marker for

melanoma (6), with increasing serum levels predictive of dis-ease stage, increased cancer recurrence, and low patient sur-vival (7, 8).

In addition to being a tumor marker, there is evidence thatS100B contributes to cancer progression via Ca2�-dependenttarget interactions that regulate cell growth and survival (6,9 –13). Specifically, each subunit of dimeric S100B has two EF-hand Ca2�-binding domains, with the C-terminal EF-handserving as a “Ca2�-activated switch” that exposes a protein tar-get-binding site on its surface (14 –18). S100B targets includeSrc kinase, serine/threonine protein kinase Ndr, E3 ubiquitinligase Hdm2, its homolog Hdm4, and, most notably, the tetra-meric p53 tumor suppressor protein (19). In the case of p53,Ca2�-dependent binding of S100B disrupts p53 oligomeriza-tion (17) and significantly lowers p53 protein levels in malig-nant melanoma (10, 11). In addition, S100B-p53 complex for-mation inhibits p53 binding to DNA, its transcriptionactivation activity, and p53-dependent apoptosis (9, 10, 15, 16,20). As mentioned, the p53 regulatory proteins Hdm2 andHdm4 are also S100B targets (17, 21). However, a detailedmechanism for how the Ca2�-dependent S100B-p53, S100B-Hdm2, and S100B-Hdm4 complexes contribute to loweringp53 levels in melanoma is not yet fully resolved (17).

MAPK is another growth signal activated by S100B, althoughthis effect is indirect and via receptor-mediated processes (19,22). The role of intracellular S100B on MAPK signaling is notwell defined, and no such information is available for melanoma(23–29). Nonetheless, ERK is overly active in 90% of melanomapatients because of activating mutations in upstream kinasessuch as BRAF (mutations in 50 –70% of melanomas) and NRAS(mutations in 15–30% of melanomas), so a myriad of ERK1/2targets involved in cell proliferation, differentiation, increased

* This works was supported, in whole or in part, by National Institutes ofHealth grants GM58888 and CA107331 (to D. J. W.) and CA166576 (toM. I. V.).

1 Both authors contributed equally to this work.2 To whom correspondence may be addressed: Dept. of Biochemistry and

Molecular Biology, 108 N. Greene St., Baltimore, MD 21201. Tel.: 410-706-6380; Fax: 410-706-0458; E-mail: [email protected].

3 To whom correspondence may be addressed: Dept. of Biochemistry andMolecular Biology, 108 N. Greene St., Baltimore, MD 21201. Tel.: 410-706-4354; Fax: 410-706-0458; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 18, pp. 12886 –12895, May 2, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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survival, and the reduction of apoptosis are impacted in mela-noma (22, 30 –32). In this study, the role of S100B on MAPKsignaling pathways was evaluated and found to directly affectRSK phosphorylation.

The RSKs4 (90-kDa ribosomal S6 kinases) represent oneimportant set of ERK1/2 substrates with four family members(RSK1– 4), each containing six highly conserved phosphoryla-tion sites (Ser-221, Thr-359, Ser-363, Thr-573, Ser-380, andSer-749) (Fig. 4A). In quiescent cells, ERK1/2 associates with adocking domain in the extreme C terminus of RSK (consensussequence LXPXXXSXLAXRRXXK) (33). Upon phorbol ester-or growth factor-dependent activation, phosphorylation ofRSK is achieved by ERK1/2, PDK1, and two autophosphoryla-tion events (34). In the canonical RSK activation pathway, ERKphosphorylates RSK in its linker region (Thr-359, Ser-363) andat Thr-573 in the C-terminal kinase domain (CTKD). The ERK-dependent phosphorylation of RSK at Thr-573 enables auto-phosphorylation at Ser-380 via residues from the CTKD. Phos-phorylated Ser-380 on RSK also creates a docking site for PDK1that, in turn, phosphorylates Ser-221 in the RSK N-terminalkinase domain. The N-terminal kinase domain of RSK is thenable to trans activate a variety of downstream protein targetsnecessary for cellular function. As part of a feedback loop, theactivated N-terminal kinase domain catalyzes a second auto-phosphorylation event within RSK at Ser-749 to dissociate theERK-RSK complex and disengage the RSK signaling cascade(35).

To better understand how elevated S100B affects ERK/RSK-mediated signaling in malignant melanoma, S100B knockdownand overexpression studies were completed using the 501mel(low S100B) and WM115 (elevated S100B) melanoma cell lines.It is generally assumed that active ERK inevitably results in thephosphorylation and activation of its downstream target p90RSKs (34, 36). However, surprisingly, inhibition of a criticalERK phosphorylation site on RSK, at Thr-573, was observedwhen S100B levels are elevated. Furthermore, phosphorylationof RSK Thr-573 was blocked via a direct Ca2�-dependent inter-action between S100B and the CTKD of RSK and promotedRSK sequestration to the cytoplasm. Thus, in addition torepressing the p53-tumor suppressor pathway (9 –11), elevatedS100B alters MAPK signaling in malignant melanoma via adirect and Ca2�-dependent interaction with RSK.

EXPERIMENTAL PROCEDURES

Cell Lines and Cell Culture—WM115 (ATCC) malignantmelanoma cells were cultured in minimal essential medium(Invitrogen) supplemented with 10% heat-inactivated FBS and100 units/ml penicillin/streptomycin. 501mel (Dr. Ruth Hala-ban, Yale University) malignant melanoma cells were culturedin RPMI medium (Invitrogen) supplemented with 10% heat-inactivated FBS and 100 units/ml penicillin/streptomycin.Where indicated, assay medium was composed of minimal

essential medium or RPMI medium with 1% charcoal-stripped,dextran-treated FBS (Hyclone) and 100 units/ml penicillin/streptomycin. All cells were maintained in a 37 °C incubatorwith 5% CO2. Both cell lines were sequenced to establish BRAFstatus. The WM115 cells harbor the activating BRAF V600Dmutation and wild-type NRAS, and the 501mel cells have WTBRAF and the activating NRAS G12D mutation (37). As shownhere by Western blot analysis, the WM115 cells have elevatedlevels of endogenous S100B protein, and the 501mel cells havelittle, if any, detectable S100B protein, which is rare in malig-nant melanoma.

Lentiviral shRNA Particle Infections—WM115 cells wereseeded in triplicate at 1 � 104 cells/well in 96-well plates innormal growth medium and allowed to recover overnight. Thecells were infected with SMARTvector 2.0 lentiviral particlescontaining either non-targeting scrambled or anti-S100BshRNA according to the recommendations of the manufac-turer (Thermo Scientific Dharmacon). The following day,the medium containing lentivirus was removed, the cellswere washed twice with PBS and trypsinized, and each wellwas expanded into a 24-well plate containing growthmedium supplemented with puromycin (0.5 �g/ml). Uponconfluence, the wells were trypsinized and single cell-dilutedinto 96-well plates. Positive clones, having significantlyreduced S100B expression, were maintained in puromycin-containing medium.

Site-directed mutagenesis—Human S100B cDNA was pur-chased from the ATCC, subcloned into the mammalian expres-sion vector pcDNA3.1(�) (Invitrogen), and confirmed bysequence analysis. Using the QuikChange II site-directedmutagenesis kit (Agilent), two successive point mutations wereintroduced into the calcium-binding domains of the S100BcDNA. First, the glutamate at position 31 was changed to analanine, and then the glutamate at position 72 was changed toan alanine, creating the E31A/E72A double mutant of S100B,and the mutations were verified by sequencing. The E31A/E72A mutations did not affect protein structure but abolisheddetectable calcium-binding activity (18).

Transfections—501mel cells were seeded in 6-well plates at2.5 � 105 cells/well and allowed to recover overnight. The501mel cells were then transfected with the pcDNA3.1(�) vec-tor alone, with the same vector containing human wild-typeS100B, or with the vector harboring the E31A/E72A doublemutant of S100B, which no longer binds Ca2� ions. The trans-fections were completed using the Mirus TransIT-LT1 reagent(Mirus Bio), following the protocol of the manufacturer. After48 h of incubation at 37 °C, the transfected cells were trans-ferred to growth medium containing neomycin (0.5 mg/ml,Invitrogen) and single cell-diluted into 96-well plates. Positiveclones overexpressing wild-type or mutant S100B were main-tained in neomycin-containing medium.

Western Blot Analysis—Western blot analyses were per-formed as described previously (38). Primary antibodies forS100B (BD Biosciences), pERK1/2, ERK1/2, pRSK1/3 (Thr-359/Ser-363), pRSK1/2/3 (Ser-380), pRSK1/2/3 (Thr-573),RSK1/2/3 (Cell Signaling Technology), and GAPDH (Calbi-ochem) were used at the dilutions recommended by the manu-facturer. Protein-antibody complexes were detected using

4 The abbreviations used are: RSK, ribosomal S6 kinase; pERK, phosphorylatedERK; CTKD, C-terminal kinase domain; BRAF, B-rapidly activated fibrosar-coma; NRAS, neuroblastoma Ras oncogene gene product; RAGE, receptorfor advanced glycation end products; BAD, Bcl-2-associated death pro-moter protein; DAPK, death-associated protein kinase.

S100B-dependent Regulation of RSK

MAY 2, 2014 • VOLUME 289 • NUMBER 18 JOURNAL OF BIOLOGICAL CHEMISTRY 12887


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Amersham Biosciences ECL Western blot detection reagents(GE Healthcare). UO126 and EGF were purchased from CellSignaling Technology and Invitrogen, respectively.

Cell Viability Assays—Cells were seeded in triplicate at 3 �103 cells/well in 96-well plates in assay medium overnight. Themedium was then changed to fresh normal growth medium(day 0). At each time point, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) was added for 3 h. Theresultant formazan crystals were solubilized by the addition of a1:10 solution of 0.1 M glycine (pH 10.5):dimethyl sulfoxide over-night at room temperature, and the absorbance (450 nm) wasmeasured. Absorbance readings at day 0 were taken to establishstarting cell viability, from which the percentage of cell viabilityover initial cells plated was calculated.

Preparation of Recombinant Proteins—The pET11b expres-sion vector (Novagen) containing the wild-type rat S100B geneor the E31A/E72A double mutant rat S100B gene was used toproduce recombinant S100B in HMS174 (DE3) cells (Nova-gen). S100B was prepared and purified (�99%) under reducingconditions using procedures similar to those described previ-ously (14, 39), except that DTT was used as a reducing agentinstead of �-mercaptoethanol. The cDNA (Addgene) for theCTKD (residues 410 –735) of human RSK1 was subcloned intothe pET-41 Ek/LIC expression vector (Novagen) according tothe recommendations of the manufacturer, and its sequencewas verified. The recombinant GST-RSK1410 –735 fusion pro-tein was expressed in Rosetta (DE3) cells (Novagen) and iso-lated using glutathione-Sepharose 4B beads (GE HealthcareBioscience) batch purification according to the instructions ofthe vendor. The same protocol was used to purify GST for useas a control. The concentrations of the protein stock solutionswere determined using a protein assay (Bio-Rad) using wild-type S100B of known concentration as the standard. The con-centration of this S100B standard was determined by quantita-tive amino acid analysis (BioSynthesis).

Kinase Assays—Active ERK2 (2 ng, Millipore) was incubatedfor 15 min with 0.5 �g of purified GST-RSK1410 –735 (�99%) at30 °C in a buffer optimized for protein kinase reactions (NewEngland Biolabs, termed NE buffer) in the presence andabsence of 0.5 �g of pure S100B (�99%). The final conditions inthe reaction buffer were 20 �M ATP, 50 mM Tris-HCl, 10 mM

MgCl2, 0.1 mM EDTA, 2.0 mM DTT, 0.01% Brij 35 (pH 7.5), and,where indicated, 1.0 mM CaCl2. The kinase reactions werestopped by adding an equal volume of 4�-concentrated SDS-PAGE sample buffer prior to analysis by Western blot.

Pulldown Assays—S100B or BSA was covalently attached tomagnetic Dynabeads according to the recommendations of themanufacturer (Invitrogen). An aliquot of protein-bound beadswas washed three times in radioimmune precipitation assaybuffer (40 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.3% Tri-ton X-100) supplemented with 5 mM EDTA or 5 mM calciumchloride, mixed with 500 �g of WM115 cell lysate for 1 h at 4 °C,and then washed again three times with radioimmune precipi-tation assay buffer. Target proteins were identified using West-ern blot analysis after boiling the beads in SDS-PAGE buffer.GST pulldown experiments were completed at 4 °C by firstwashing glutathione-Sepharose 4B beads (100 �l, GE Health-care Bioscience) three times with TBS containing either 5 mM

EDTA or 5 mM calcium chloride and then incubating the pre-washed beads with 200 �g of GST or GST-RSK1410 –735 for 30min. Next, the protein-bead complexes were washed threetimes and blocked with 1% BSA in TBS for 30 min, and thenpurified S100B protein (500 �g) or the E31A/E72A mutantS100B protein (500 �g) was added, rocked for 2 h at 4 °C, andwashed five times. The beads were boiled in SDS-PAGE buffer,and the eluted proteins were analyzed by Western blot analysis.

Subcellular Fractionation—Cells were grown and harvestedat subconfluence, cell pellets were prepared for cytoplasmicand nuclear extraction using the NucBuster kit (Novagen)according to the recommendations of the manufacturer, andthe subcellular fractions were analyzed by Western blot analy-sis. Primary antibodies for MEK1/2 (Cell Signaling Technol-ogy) and p84 (Abcam) served as controls for cytoplasmic andnuclear fractions, respectively, and were prepared using dilu-tions recommended by the manufacturer.

Immunofluorescence—Cells were grown on glass coverslips,fixed with methanol, and blocked for 1 h in 10 mg/ml BSA and0.1% Tween 20 in PBS at room temperature. S100B and pRSK573 were stained using anti-S100B antibody (1:500, BD Biosci-ences) and anti-pRSK 573 (1:400, Cell Signaling Technology),followed by Alexa Fluor 488- and Alexa Fluor 568-conjugatedsecondary antibody (1:1000, Invitrogen) and Hoechst 33342(1:5000, Sigma). Z-stacks were obtained using an OlympusFV1000 confocal microscope. Stacks were split for maximumintensity projections using ImageJ.

RESULTS

Elevated S100B Levels Positively Correlate with IncreasedMelanoma Cell Viability—The role of S100B in melanoma pro-gression has been established previously (10, 11). However, allmelanoma cells tested already expressed elevated S100B.Therefore, to elucidate whether S100B promotes melanomaviability, it was important to examine a melanoma cell line withlow levels of S100B and determine the effect of adding S100B.This is particularly important because patients with low levelsof S100B generally have a better prognosis in the clinic andoften benefit from malignant melanoma immunotherapyapproaches. The 501mel cells were critical for these studiesbecause they do not express detectable levels of S100B, which isa rarity for human malignant melanoma (40, 41). For thesestudies, separate single cell-derived S100B overexpression lineswere derived from 501mel cells, and elevated S100B proteinexpression was confirmed by Western blot analyses (Fig. 1B).After 7 days, a significant increase in cell viability was observedfor the S100B-expressing cells compared with cells containingthe empty vector alone. In complementary experiments, stableshRNA expression was used to block S100B expression inWM115 melanoma cells that express high levels of endogenousS100B (Fig. 1A). For these studies, WM115 cells were infectedwith lentivirus containing human anti-S100B shRNA, and sin-gle cell clones were derived. Multiple clones were examined byWestern blot analysis, and significant knockdown of S100Bprotein was observed (�99%). As a control, WM115 cells wereinfected with lentivirus containing scrambled non-targetingshRNA. After verifying significant S100B expression knock-down, cell viability was determined by 3-(4,5-dimethylthiazol-




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2-yl)-2,5-diphenyltetrazolium bromide assays. Both S100Bknockdown clones 1 and 2 had decreased cell viability after 7days compared with control cells with elevated S100B (Fig. 1A).The S100B knockdown and overexpression studies demon-strate that higher levels of S100B, as found in most malignantmelanoma patients, directly correlate with increased cellularviability. The cell viability results from these cells lines are inagreement with previous S100B knockdown studies performedin human C8146A cells that were derived from patients withmalignant melanoma (9 –11).

S100B Inhibits ERK-dependent Phosphorylation of RSK atResidue Thr-573—One potential mechanism for affecting cellviability is an increased activation of the MAPK signaling cas-

cade. Although the effects of S100B on phosphorylation andactivation of ERK (i.e. to pERK) via cell-surface receptors is wellestablished in several cell types (23–29), little is known abouthow intracellular S100B affects MAPK signaling, particularly inmalignant melanoma where S100B levels are typically elevated.Therefore, the effect S100B expression has in 501mel or itsinhibition in WM115 cells was examined first with respect toERK activity. When intracellular S100B expression was blockedin WM115 cells, either no change or a small decrease in pERKwas observed, whereas an increase in pERK was never seen (Fig.2A). Consistent with the knockdown experiments but unlikerobust increases in phosphorylated ERK (pERK) from receptor-mediated responses, a large increase in activated ERK was notobserved with increased S100B in 501mel malignant melanoma

FIGURE 1. S100B increases melanoma cell viability. A, Western blot analysisof total cell lysates (25 �g) showing the level of S100B expression of thenon-targeting scrambled WM115 cells (lane S) as well as two S100B knock-down clonal lines (inset, lanes 1 and 2). Cellular viability of the knockdown cellscompared with that of the non-targeting scrambled cells was assessed atseveral time points over the course of 7 days using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay to determine thechange in cell number. The scrambled control cells (S), S100B knockdownclone 1 (1), and clone 2 (2) are also represented as in the inset. Shown is acompilation of three different experiments, each performed in triplicate (n �9). B, Western blot analysis was performed on total cell lysate (25 �g) to con-firm expression of S100B in the 501mel cells. Shown are 501mel cells contain-ing vector alone (V) and the S100B clonal lines A and B (inset). Cellular viabilityof the 501mel S100B-expressing clonal lines was compared with that of thevector control cells at several time points measured over a period of 7 days.The vector control cells (V), S100B-expressing clone A (A), and clone B (B) arerepresented as in the inset. Shown is a compilation of three different experi-ments, each performed in triplicate (n � 9).

FIGURE 2. S100B suppresses RSK Thr-573 phosphorylation by ERK. A,Western blot analysis (25 �g) of WM115 cells with the non-targeting scram-bled vector (S) and two stable S100B knockdown clonal cell lines derived fromWM115 cells (1 and 2). B, Western blot analysis (20 �g) showing the effects ofS100B expression in 501mel cells (first and second lanes) compared with anempty vector control (third and fourth lanes) in the absence (first and thirdlanes) or presence (second and fourth lanes) of the MEK1/2 inhibitor (5 �g/ml)U0126.




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cells (Fig. 3B). Thus, no reductions of phosphorylated ERK wereobserved, and the apparent slight increases in some experi-ments were likely experimental variability (Figs. 3B and 5A).These data indicate that intracellular S100B levels increaseminimally or have no effect on ERK phosphorylation in thesetwo melanoma cell lines.

However, despite finding that intracellular S100B minimallyaffects pERK levels, if at all, we discovered that phosphorylationof RSK, a downstream target of ERK, was inhibited significantlywhen S100B levels were increased (Fig. 6). Specifically, the sta-ble introduction of S100B into 501mel malignant melanomacells inhibited the phosphorylation of RSK at Thr-573 (Figs. 2Band 3B) without affecting the phosphorylation of other ERK-dependent sites on RSK such as Thr-359/Ser-363 (Figs. 2B and3B). The ability to block one ERK site on RSK suggests thatS100B interacts with RSK directly. Previously, S100B has beenshown to interact with other kinase substrates, rather than thekinase itself, to block phosphorylation via steric and/or allos-teric mechanisms (16). A S100B binding site within the CTKD

of RSK, sterically blocking Thr-573, would be consistent withthis mechanism.

To confirm that ERK is indeed responsible for the phosphor-ylation of RSK in these cells, MEK1/2 was inhibited with U0126(Fig. 2B). The addition of U0126 was found to abolish ERKphosphorylation in the 50lmel cells. Likewise, a drastic decreasein RSK phosphorylation occurred at Thr-573, Thr-359/Ser-363, and Ser-380 and demonstrated that ERK is indeed respon-sible for the majority of RSK phosphorylation in 501mel cells.As an aside, a very low level of ERK-independent phosphoryla-tion of RSK was observed (Fig. 3), but this residual phosphoryl-ation was not affected by S100B addition (data not shown). In acomplementary experiment, EGF was added to serum-starvedWM115. However, pERK levels did not decrease with serumstarvation, and EGF addition did not significantly increasepERK (data not shown). Thus, changing pERK levels in WM115cells could not be easily achieved, which is not too surprisingbecause WM115 cells already have activated ERK from theoncogenic BRAF mutation (i.e. V600E). As a result, the dynamicrange for pERK levels is quite small, and the resulting effectsfrom exogenous growth factors are minimal, if observed at all,as described previously (42).

The S100B Inhibitory Effect on RSK Phosphorylation IsCa2�-dependent—To evaluate whether S100B protein alone issufficient to inhibit ERK-dependent phosphorylation of RSK,cell-free protein kinase assays were completed. In these studies,ERK2-mediated phosphorylation of the RSK C-terminal kinasedomain (RSK1410 –735) at Thr-573 was monitored in theabsence and presence of S100B (Fig. 3A). ERK2 successfullycatalyzed the phosphorylation of RSK1410 –735 within 15 min ofincubation. In the presence of S100B and Ca2�, RSK phosphor-ylation by ERK2 was decreased significantly. However, thiseffect was abolished in the presence of a Ca2� chelator, EDTA(Fig. 3A). These data show that S100B inhibits ERK-dependentphosphorylation of RSK at Thr-573 in a Ca2�-dependentmanner.

To further prove whether Ca2� was necessary for S100B toexert its inhibitory effect on RSK Thr-573 phosphorylation incells, an E31A/E72A double mutant of S100B was tested, whichis incapable of Ca2� binding. A comparison of protein levelsover time using Western blot analysis confirmed that the sta-bility of S100B within 501mel cells was not affected by the twomutations (data not shown). The S100B double mutant wassimilarly found to have no effect on activated ERK levels, and ithad no effect on ERK-dependent phosphorylation of RSK atThr-573 (Fig. 3B). These data provided additional evidence thatthe Ca2�-binding properties of S100B are necessary for itsmodulatory effects on RSK. Importantly, for the first time, thesedata also show the Ca2� requirement for an S100B functionwithin cells.

S100B Binds to RSK in a Ca2�-dependent Manner—Protein-protein interactions involving S100B require a Ca2�-depen-dent conformational change to expose its protein target-bind-ing site (14). Therefore, binding experiments were performedin vitro to determine whether S100B binds to ERK and/or toRSK directly (Fig. 4). For these studies, S100B or BSA was cova-lently attached to magnetic Dynabeads and mixed withWM115 cell lysate in the absence (EDTA-treated) or presence

FIGURE 3. Inhibition of RSK phosphorylation by S100B is Ca2�-depen-dent. A, in vitro kinase assays examining the ERK-mediated phosphorylationof RSK Thr-573 in the absence and presence of S100B and 1 mM CaCl2. Westernblot analysis was employed following a 15-min incubation period, andchanges in pRSK at Thr-573 were observed. B, Western blot analysis of 25 �gof 501mel cell lysate with untreated cells (first lane) or cells transfected withempty vector (second lane), a vector with wild-type S100B (third lane), or avector with the E31A/E72A double mutant of S100B (fourth lane), which is anS100B construct incapable of binding calcium (18). These data confirm thatS100B must bind calcium ions to bind and inhibit RSK phosphorylation atThr-573.




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of Ca2�. Western blot analysis of the eluates revealed that full-length cellular RSK, but not full-length ERK, bound to S100B ina Ca2�-dependent manner (Fig. 4B). Because S100B blockedthe phosphorylation of several protein kinase C substrates bysterically preventing kinase access to the phosphorylation site,rather than by inhibiting kinase enzymatic activity (16, 20), theability of S100B to bind to the CTKD of RSK, containing Thr-573, was then tested. These binding experiments were per-formed with a GST-tagged RSK410 –735 construct with eitherpurified recombinant WT S100B or the Ca2�-binding doublemutant (E31A/E72A, Fig. 4C). It is important to note that theE31A/E72A double mutant was sufficient to fully abolish Ca2�-binding (KD� 500 mM) without affecting the overall structureof the protein, as evaluated by protein NMR (18). In this seriesof binding experiments, analysis of the eluates showed that WTS100B, but not the double mutant, bound to GST-RSK1410 –735.To further prove that the binding of S100B to the CTKD of RSKis Ca2�-dependent, binding was evaluated in the absence andpresence of the Ca2� chelator EDTA (Fig. 4). Together, thesedata demonstrated that S100B binds directly within the CTKDof RSK and that S100B binding to the CTKD of RSK is Ca2�-

dependent. Because recombinant proteins were used to studythis S100B-RSK1410 –735 complex, no posttranslational modifi-cations were necessary for this protein-protein interaction.

Elevated S100B Inhibits the Nuclear Localization of RSK—RSK resides in the cytoplasm until mitogenic activation causesits translocation into the nucleus via an unknown mechanism(43). As a result, RSK is capable of phosphorylating differentprotein targets, depending on its cellular localization (36). Wenext determined whether S100B affected RSK cellular localiza-tion. Cytoplasmic and nuclear protein fractions were isolatedfrom WM115 cells expressing non-targeting scrambled or anti-S100B shRNA. Western blot analyses showed an increase innuclear RSK and phosphorylated Thr-573 RSK (pRSK573) inthe S100B knockdown clone in comparison with control cells(Fig. 5A). Immunofluorescence studies showed that pRSK573 isenriched in the nuclei of S100B knockdown cells and that con-trol vectors with elevated S100B showed diffuse staining ofpRSK573 in the cytoplasm (Fig. 5B). To be certain, confocalXYZ image stacks established that pRSK573 is cytoplasmic andis above or below the nucleus. Areas within the nuclear bound-aries that are devoid of DNA staining are typical of nucleoli, andthe exclusion of pRSK573 from these areas is consistent withboth a nuclear localization for pRSK573 and previous observa-tions that RSK does not typically accumulate in nucleoli (Fig.5C) (44). In summary, elevated S100B contributes to diffusecytoplasmic staining of RSK, whereas blocking S100B expres-sion allows for RSK translocations into the nucleus as necessaryfor the nuclear component of its biological activities (Fig. 6).These studies indicate that S100B is able to block RSK nuclearlocalization.

DISCUSSION

Increased S100B levels contribute to cancer cell growth andsurvival as shown here (Fig. 1) and elsewhere (19). One poten-tial mechanism for S100B-dependent cell growth is its ability toactivate ERK in an indirect manner via cell-surface receptorssuch as RAGE (23–29). However, other explanations can beprovided. These include intracellular S100B-target interactionssuch as those involved in inhibiting p53 activities (i.e. S100B-p53, S100B-hdm2, and S100B-hdm4) (9 –11) and/or from otherS100B-target interactions reviewed elsewhere (19). In thisstudy, we examined, for the first time, whether intracellularS100B had a direct effect on MAPK signaling in malignant mel-anoma. Although no direct interaction was found betweenS100B and ERK, a Ca2�-dependent interaction was detectedbetween S100B and a downstream ERK target, RSK (Fig. 4). Asa result of this complex, S100B uniquely modulated a down-stream ERK signal by blocking the phosphorylation of RSK atThr-573 and preventing its nuclear localization (Figs. 2, 3, and5). Such an effect would thereby inhibit the effects of RSK on itsnuclear targets, and possibly increase its activity toward cyto-plasmic targets (Fig. 6). The binding of S100B and RSK werealso shown to be dependent on Ca2�, and, thus, S100B links twoimportant signaling pathways involved in regulating cellgrowth/survival (i.e. MAPK and Ca2� signaling). Finally, theresults presented here answer important but previously unre-solved questions about the ability of S100B to bind intracellular

FIGURE 4. S100B directly binds to RSK and a construct of RSK with theCTKD but not to ERK. A, schematic of RSK1 (residues 1–735) showing theN-terminal kinase domain (NTKD, residues 62–321), the CTKD (residues 418 –675), the ERK binding site at the C terminus of RSK1 (residues 722–735, under-lined), and the phosphorylation sites reported to be necessary for RSK activa-tion are labeled P (43). B, Western blot analysis of RSK and ERK protein levelsfrom BSA or S100B pulldown eluates performed in the presence of either 5mM EDTA (first through third lanes) or 5 mM CaCl2 (fourth through sixth lanes).The first and fourth lanes contain total WM115 cell lysate antibody controls(CT), whereas eluates of BSA and lysate are shown in the second and fifth lanes,and S100B and lysate are shown in the third and sixth lanes. C, Western blotanalysis of GST or GST-RSK1386 –752 cell-free pulldowns supplemented witheither 5 mM EDTA (second through fifth lanes) or 5 mM calcium (sixth throughninth lanes). Eluates of GST with S100B (second and sixth lanes), GST-RSK1386 –

752 with S100B (third and seventh lanes), GST with the E31A/E72A mutant(fourth and eighth lanes), and GST-RSK1386 –752 with the E31A/E72A mutant(fifth and ninth lanes) are shown. S100B protein was loaded in the first lane asan antibody control.




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FIGURE 5. RSK nuclear localization is inhibited by S100B. A, Western blot analysis comparing the cytoplasmic (C) and nuclear (N) protein levels of S100Bknockdown clone 1 (1) to those of the non-targeting scrambled cell line (S). Shown is an example experiment that was repeated in triplicate. The nuclear matrixprotein p84 and MEK were used as loading controls for the nuclear and cytoplasmic fractions, respectively. B, immunofluorescence studies showing thelocalization of pRSK Thr-573 in both the S100B knockdown clone 1 and non-targeting scrambled cell lines (representative of three different fields of vision areshown). C, immunofluorescence studies focusing on a single XYZ image to show the localization of pRSK Thr-573 as well as nucleolar exclusion for each cell line.Planes are indicated by dotted lines and examples of nucleolar exclusion by arrows (representative of numerous XYZ images).

FIGURE 6. Schematic representation of the Ca2�-dependent effects of S100B on the MAPK signaling cascade. S100B-Ca2� directly interacts with RSK,inhibiting phosphorylation at Thr-573 by ERK and reducing subsequent translocation of RSK to the nucleus, thus allowing it to act on its cytoplasmic targets butnot on nuclear targets. Phosphorylation sites on RSK are labeled with the letter P.




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Ca2� ions and challenge current models for ERK-dependentactivation of RSK.

The S100B-dependent inhibition of RSK Thr-573 phosphor-ylation by ERK in the cell does not occur with the Ca2�-bindingmutant (Figs. 3 and 4), demonstrating that S100B undoubtedlybinds Ca2� ions inside the cell. This is important because theCa2�-binding affinities reported in vitro for the “typical” EF-hand and the “pseudo” EF-hand of S100B are KD �20 �M and�200 �M, respectively (45). Therefore, it has been questionedby many as to whether S100B could be an active signaling pro-tein inside the cell where physiological Ca2� ion concentrationsare generally very low (0.1–2 �M) (46). It is possible that localCa2� concentration gradients exist and/or increased Ca2� lev-els occur in cancer cells because of aberrant regulation allowingS100B to be activated (47). However, another explanation isthat the Ca2� binding affinity of S100 proteins can also beincreased upon binding other metals and/or their physiologi-cally relevant protein target(s). For example, it is well estab-lished in vitro that the affinity of S100B and other S100 proteinsfor Ca2� is increased after Zn2� binding (48, 49), redox modi-fication of critical cysteine residues (50), and/or by target bind-ing, although the mechanism by which Ca2� binding isincreased by as much as 200-fold upon target binding to S100Bis still under investigation (18, 51–53).

Another finding reported here is that increased S100B inhib-ited ERK-dependent phosphorylation of RSK at residue Thr-573 via a direct and Ca2�-dependent interaction (Fig. 6). In thesame study, S100B had no effect on the phosphorylation ofresidues Thr-359/Ser-363 or Ser-380 of RSK (Figs. 2B and 3B).This is in conflict with the current model of RSK activation bysequential phosphorylation (43), where it is thought that ERKphosphorylation of Thr-573 occurs first, followed by ERK phos-phorylation of Thr-359/Ser-363 in the linker region and theCTKD-mediated autophosphorylation of Ser-380. pSer-380 issaid to create a binding site for PDK1, which phosphorylatesRSK Ser-221, to fully activate RSK. However, because Thr-573phosphorylation is blocked by S100B, we have shown that phos-phorylation of Thr-573 is not required for RSK CTKD auto-phosphorylation of Ser-380. The ERK-dependent phosphory-lation of RSK Thr-359/Ser-363 is sufficient to phosphorylatethe remaining sequential sites on RSK, although it remains pos-sible that an alternative kinase is responsible. The effects ofS100B may not be limited to RSK, so additional studies areongoing to determine whether S100B selectively inhibits thephosphorylation of other substrates of ERK.

The Ca2�-dependent binding of S100B to RSK was nextfound to block RSK nuclear localization (Fig. 5). Because RSKhas specific functionality in the cytoplasm and the nucleus, itsrestriction to the cytosol in melanoma cells may preferentiallydrive the activation of only a subset of RSK targets. Likewise, thesequestering of RSK in the cytosol would prevent it from per-forming any necessary nuclear functions (Fig. 6). Although wehave already shown that S100B inhibition of p53 plays a role inincreased melanoma survival (9, 10, 15, 16, 20), modification incytosolic and nuclear signaling because of S100B binding ofRSK and the corresponding change in RSK localization couldalso contribute to increased cell proliferation and survival. Forexample, in the cytoplasm, RSK could contribute to increased

cell growth by inducing the degradation of the NF-�B inhibitorI�B� (54 –56), suppressing BAD-mediated apoptosis (57, 58)and/or inactivating tumor suppressors such as DAPK andTSC2 (43, 59). Also, a recent study showed that MAPK-acti-vated RSK promotes melanoma growth by increasing the activ-ity of another cytoplasmic target, mammalian target of rapamy-cin complex 1, which is known to regulate cell growth, cellproliferation, cell motility, cell survival, protein synthesis, andtranscription (35, 60). In contrast, the sequestration of S100B ofRSK to the cytoplasm would decrease the activity of numeroustarget transcription factors, several of which promote differen-tiation (35, 61). Future experimentation is certainly required toinvestigate these and numerous other hypotheses stemmingfrom these findings involving S100B-dependent regulation ofMAPK signaling.

How S100B sequesters RSK in the cytoplasm is not known. Itmay sterically block its entry into the nucleus, mask a nuclearlocalization signal on RSK, and/or require Thr-573 phosphory-lation for nuclear localization. Similarly, the small death effec-tor domain protein PEA-15 (phosphoprotein enriched in astro-cytes, 15 kDa) binds RSK2 and prevents it from entering thenucleus (62). The authors of the RSK2 study did not note anychange in the phosphorylation state of RSK2, suggesting simplythat its interaction with PEA-15 alone was enough to sequesterit to the cytoplasm. Unlike PEA-15, the S100B interaction withRSK is Ca2�-regulated and, thus, links Ca2� and RSK signaling.A possible mechanism linking MAPK and Ca2� signaling is thatat elevated Ca2� levels, RSK binds Ca2�-S100B and is seques-tered to the cytoplasm, where it is able to phosphorylate a cer-tain subset of target substrates, whereas, at lower levels of Ca2�,RSK is not bound to S100B and can freely translocate to thenucleus, modulating its nuclear targets (Fig. 6).

Like other Ca2�-signaling proteins (i.e. calmodulin, troponinC, etc.), S100 proteins regulate multiple biological activities.However, unlike calmodulin, which is ubiquitously expressed,the 24 S100 family members regulate individual biologicalactivities in a cell-specific manner (12, 13, 19, 63, 64). It has alsobeen established that S100B levels are highly elevated in malig-nant melanoma (3). However, our understanding of how S100Bcontributes to the melanoma phenotype is not fully under-stood. In this study, the effects of varying S100B levels onMAPK signaling were examined, providing more evidence thatS100B is a direct mediator of tumor survival and not just aprognostic marker in this deadly cancer.

REFERENCES1. Zimmer, D. B., Cornwall, E. H., Landar, A., and Song, W. (1995) The S100

protein family: history, function, and expression. Brain Res. Bull. 37,417– 429

2. Donato, R. (2001) S100: a multigenic family of calcium-modulated pro-teins of the EF-hand type with intracellular and extracellular functionalroles. Int. J. Biochem. Cell Biol. 33, 637– 668

3. Gaynor, R., Herschman, H. R., Irie, R., Jones, P., Morton, D., and Cochran,A. (1981) S100 protein: a marker for human malignant melanomas? Lan-cet 1, 869 – 871

4. Nagasaka, A., Umekawa, H., Hidaka, H., Iwase, K., Nakai, A., Ariyoshi, Y.,Ohyama, T., Aono, T., Nakagawa, H., and Ohtani, S. (1987) Increase inS-100b protein content in thyroid carcinoma. Metabolism 36, 388 –391

5. Yang, J. F., Zhang, X. Y., and Qi, F. (2004) Expression of S100 protein inrenal cell carcinoma and its relation with p53. Zhong Nan. Da Xue Xue




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Bao. Yi Xue. Ban 29, 301–3046. Zimmer, D. B., Lapidus, R. G., and Weber, D. J. (2013) In vivo screening of

S100B inhibitors for melanoma therapy. Methods Mol. Biol. 963, 303–3177. Harpio, R., and Einarsson, R. (2004) S100 proteins as cancer biomarkers

with focus on S100B in malignant melanoma. Clin. Biochem. 37, 512–5188. Hauschild, A., Engel, G., Brenner, W., Gläser, R., Mönig, H., Henze, E., and

Christophers, E. (1999) S100B protein detection in serum is a significantprognostic factor in metastatic melanoma. Oncology 56, 338 –344

9. Lin, J., Blake, M., Tang, C., Zimmer, D., Rustandi, R. R., Weber, D. J., andCarrier, F. (2001) Inhibition of p53 transcriptional activity by the S100Bcalcium-binding protein. J. Biol. Chem. 276, 35037–35041

10. Lin, J., Yang, Q., Wilder, P. T., Carrier, F., and Weber, D. J. (2010) Thecalcium-binding protein S100B down-regulates p53 and apoptosis in ma-lignant melanoma. J. Biol. Chem. 285, 27487–27498

11. Lin, J., Yang, Q., Yan, Z., Markowitz, J., Wilder, P. T., Carrier, F., andWeber, D. J. (2004) Inhibiting S100B restores p53 levels in primary malig-nant melanoma cancer cells. J. Biol. Chem. 279, 34071–34077

12. Zimmer, D. B., and Weber, D. J. (2010) The calcium-dependent interac-tion of S100B with its protein targets. Cardiovasc. Psychiatry Neurol.2010, 1–17

13. Zimmer, D. B., Wright Sadosky, P., and Weber, D. J. (2003) Molecularmechanisms of S100-target protein interactions. Microsc. Res. Tech. 60,552–559

14. Drohat, A. C., Baldisseri, D. M., Rustandi, R. R., and Weber, D. J. (1998)Solution structure of calcium-bound rat S100B(��) as determined by nu-clear magnetic resonance spectroscopy. Biochemistry 37, 2729 –2740

15. Rustandi, R. R., Drohat, A. C., Baldisseri, D. M., Wilder, P. T., and Weber,D. J. (1998) The Ca2�-dependent interaction of S100B(��) with a peptidederived from p53. Biochemistry 37, 1951–1960

16. Wilder, P. T., Rustandi, R. R., Drohat, A. C., and Weber, D. J. (1998)S100B(��) inhibits the protein kinase C-dependent phosphorylation of apeptide derived from p53 in a Ca2�-dependent manner. Protein Sci. 7,794 –798

17. Wilder, P. T., Lin, J., Bair, C. L., Charpentier, T. H., Yang, D., Liriano, M.,Varney, K. M., Lee, A., Oppenheim, A. B., Adhya, S., Carrier, F., andWeber, D. J. (2006) Recognition of the tumor suppressor protein p53 andother protein targets by the calcium-binding protein S100B. Biochim. Bio-phys. Acta 1763, 1284 –1297

18. Markowitz, J., Rustandi, R. R., Varney, K. M., Wilder, P. T., Udan, R., Wu,S. L., Horrocks, W. D., and Weber, D. J. (2005) Calcium-binding propertiesof wild-type and EF-hand mutants of S100B in the presence and absence ofa peptide derived from the C-terminal negative regulatory domain of p53.Biochemistry 44, 7305–7314

19. Donato, R., Cannon, B. R., Sorci, G., Riuzzi, F., Hsu, K., Weber, D. J., andGeczy, C. L. (2013) Functions of S100 proteins. Curr. Mol. Med. 13, 24 –57

20. Baudier, J., Delphin, C., Grunwald, D., Khochbin, S., and Lawrence, J. J.(1992) Characterization of the tumor suppressor protein p53 as a proteinkinase C substrate and a S100B-binding protein. Proc. Natl. Acad. Sci.U.S.A. 89, 11627–11631

21. van Dieck, J., Lum, J. K., Teufel, D. P., and Fersht, A. R. (2010) S100 pro-teins interact with the N-terminal domain of MDM2. FEBS Lett. 584,3269 –3274

22. Boutros, T., Chevet, E., and Metrakos, P. (2008) Mitogen-activated protein(MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth,death, and cancer. Pharmacol. Rev. 60, 261–310

23. Bianchi, R., Kastrisianaki, E., Giambanco, I., and Donato, R. (2011) S100Bprotein stimulates microglia migration via RAGE-dependent up-regula-tion of chemokine expression and release. J. Biol. Chem. 286, 7214 –7226

24. Gonçalves, D. S., Lenz, G., Karl, J., Gonçalves, C. A., and Rodnight, R.(2000) Extracellular S100B protein modulates ERK in astrocyte cultures.Neuroreport 11, 807– 809

25. Jung, D. H., Kim, Y. S., Kim, N. H., Lee, J., Jang, D. S., and Kim, J. S. (2010)Extract of Cassiae semen and its major compound inhibit S100B-inducedTGF-�1 and fibronectin expression in mouse glomerular mesangial cells.Eur. J. Pharmacol. 641, 7–14

26. Loeser, R. F., Yammani, R. R., Carlson, C. S., Chen, H., Cole, A., Im, H. J.,Bursch, L. S., and Yan, S. D. (2005) Articular chondrocytes express thereceptor for advanced glycation end products: potential role in osteoar-

thritis. Arthritis Rheum. 52, 2376 –238527. Riuzzi, F., Sorci, G., and Donato, R. (2006) S100B stimulates myoblast

proliferation and inhibits myoblast differentiation by independently stim-ulating ERK1/2 and inhibiting p38 MAPK. J. Cell Physiol. 207, 461– 470

28. Shanmugam, N., Kim, Y. S., Lanting, L., and Natarajan, R. (2003) Regula-tion of cyclooxygenase-2 expression in monocytes by ligation of the re-ceptor for advanced glycation end products. J. Biol. Chem. 278,34834 –34844

29. Tsoporis, J. N., Izhar, S., Proteau, G., Slaughter, G., and Parker, T. G. (2012)S100B-RAGE dependent VEGF secretion by cardiac myocytes inducesmyofibroblast proliferation. J. Mol. Cell Cardiol. 52, 464 – 473

30. Lopez-Bergami, P. (2011) The role of mitogen- and stress-activated pro-tein kinase pathways in melanoma. Pigment Cell Melanoma Res. 24,902–921

31. Gray-Schopfer, V., Wellbrock, C., and Marais, R. (2007) Melanoma biol-ogy and new targeted therapy. Nature 445, 851– 857

32. Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Tea-gue, J., Woffendin, H., Garnett, M. J., Bottomley, W., Davis, N., Dicks, E.,Ewing, R., Floyd, Y., Gray, K., Hall, S., Hawes, R., Hughes, J., Kosmidou, V.,Menzies, A., Mould, C., Parker, A., Stevens, C., Watt, S., Hooper, S., Wil-son, R., Jayatilake, H., Gusterson, B. A., Cooper, C., Shipley, J., Hargrave,D., Pritchard-Jones, K., Maitland, N., Chenevix-Trench, G., Riggins, G. J.,Bigner, D. D., Palmieri, G., Cossu, A., Flanagan, A., Nicholson, A., Ho,J. W., Leung, S. Y., Yuen, S. T., Weber, B. L., Seigler, H. F., Darrow, T. L.,Paterson, H., Marais, R., Marshall, C. J., Wooster, R., Stratton, M. R., andFutreal, P. A. (2002) Mutations of the BRAF gene in human cancer. Nature417, 949 –954

33. Gavin, A. C., and Nebreda, A. R. (1999) A MAP kinase docking site isrequired for phosphorylation and activation of p90(rsk)/MAPKAP ki-nase-1. Curr. Biol. 9, 281–284

34. Richards, S. A., Fu, J., Romanelli, A., Shimamura, A., and Blenis, J. (1999)Ribosomal S6 kinase 1 (RSK1) activation requires signals dependent onand independent of the MAP kinase ERK. Curr. Biol. 9, 810 – 820

35. Yang, X., Matsuda, K., Bialek, P., Jacquot, S., Masuoka, H. C., Schinke, T.,Li, L., Brancorsini, S., Sassone-Corsi, P., Townes, T. M., Hanauer, A., andKarsenty, G. (2004) ATF4 is a substrate of RSK2 and an essential regulatorof osteoblast biology: implication for Coffin-Lowry syndrome. Cell 117,387–398

36. Richards, S. A., Dreisbach, V. C., Murphy, L. O., and Blenis, J. (2001)Characterization of regulatory events associated with membrane target-ing of p90 ribosomal S6 kinase 1. Mol. Cell Biol. 21, 7470 –7480

37. Lin, W. M., Baker, A. C., Beroukhim, R., Winckler, W., Feng, W.,Marmion, J. M., Laine, E., Greulich, H., Tseng, H., Gates, C., Hodi, F. S.,Dranoff, G., Sellers, W. R., Thomas, R. K., Meyerson, M., Golub, T. R.,Dummer, R., Herlyn, M., Getz, G., and Garraway, L. A. (2008) Modelinggenomic diversity and tumor dependency in malignant melanoma. CancerRes. 68, 664 – 673

38. Vitolo, M. I., Weiss, M. B., Szmacinski, M., Tahir, K., Waldman, T., Park,B. H., Martin, S. S., Weber, D. J., and Bachman, K. E. (2009) Deletion ofPTEN promotes tumorigenic signaling, resistance to anoikis, and alteredresponse to chemotherapeutic agents in human mammary epithelial cells.Cancer Res. 69, 8275– 8283

39. Amburgey, J. C., Abildgaard, F., Starich, M. R., Shah, S., Hilt, D. C., andWeber, D. J. (1995) 1H, 13C, and 15N NMR assignments and solutionsecondary structure of rat Apo-S100 �. J. Biomol. NMR 6, 171–179

40. Böni, R., Burg, G., Doguoglu, A., Ilg, E. C., Schäfer, B. W., Müller, B., andHeizmann, C. W. (1997) Immunohistochemical localization of the Ca2�

binding S100 proteins in normal human skin and melanocytic lesions.Br. J. Dermatol. 137, 39 – 43

41. Böni, R., Heizmann, C. W., Doguoglu, A., Ilg, E. C., Schäfer, B. W., Dum-mer, R., and Burg, G. (1997) Ca2�-binding proteins S100A6 and S100B inprimary cutaneous melanoma. J. Cutan. Pathol. 24, 76 – 80

42. Bhatt, K. V., Hu, R., Spofford, L. S., and Aplin, A. E. (2007) Mutant B-RAFsignaling and cyclin D1 regulate Cks1/S-phase kinase-associated protein2-mediated degradation of p27Kip1 in human melanoma cells. Oncogene26, 1056 –1066

43. Romeo, Y., Moreau, J., Zindy, P. J., Saba-El-Leil, M., Lavoie, G., Dandachi,F., Baptissart, M., Borden, K. L., Meloche, S., and Roux, P. P. (2013) RSK




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

regulates activated BRAF signalling to mTORC1 and promotes melanomagrowth. Oncogene 32, 2917–2926

44. Chen, R. H., Sarnecki, C., and Blenis, J. (1992) Nuclear localization andregulation of Erk- and Rsk-encoded protein kinases. Mol. Cell Biol. 12,915–927

45. Chaudhuri, D., Horrocks, W. D., Jr., Amburgey, J. C., and Weber, D. J.(1997) Characterization of lanthanide ion binding to the EF-hand proteinS100 � by luminescence spectroscopy. Biochemistry 36, 9674 –9680

46. Berridge, M. J., and Irvine, R. F. (1989) Inositol phosphates and cell signal-ling. Nature 341, 197–205

47. Chen, Y. F., Chen, Y. T., Chiu, W. T., and Shen, M. R. (2013) Remodelingof calcium signaling in tumor progression. J. Biomed. Sci. 20, 23

48. Wilder, P. T., Baldisseri, D. M., Udan, R., Vallely, K. M., and Weber, D. J.(2003) Location of the Zn2�-binding site on S100B as determined by NMRspectroscopy and site-directed mutagenesis. Biochemistry 42,13410 –13421

49. Wilder, P. T., Varney, K. M., Weiss, M. B., Gitti, R. K., and Weber, D. J.(2005) Solution structure of zinc- and calcium-bound rat S100B as deter-mined by nuclear magnetic resonance spectroscopy. Biochemistry 44,5690 –5702

50. Goch, G., Vdovenko, S., Kozłowska, H., and Bierzyñski, A. (2005) Affinityof S100A1 protein for calcium increases dramatically upon glutathionyla-tion. FEBS J. 272, 2557–2565

51. Charpentier, T. H., Thompson, L. E., Liriano, M. A., Varney, K. M., Wilder,P. T., Pozharski, E., Toth, E. A., and Weber, D. J. (2010) The effects of CapZpeptide (TRTK-12) binding to S100B-Ca2� as examined by NMR andX-ray crystallography. J. Mol. Biol. 396, 1227–1243

52. Liriano, M. A., Varney, K. M., Wright, N. T., Hoffman, C. L., Toth, E. A.,Ishima, R., and Weber, D. J. (2012) Target binding to S100B reduces Dy-namic properties and increases Ca2�-binding affinity for wild type andEF-Hand mutant proteins. J. Mol. Biol. 423, 365–385

53. Wright, N. T., Prosser, B. L., Varney, K. M., Zimmer, D. B., Schneider,M. F., and Weber, D. J. (2008) S100A1 and calmodulin compete for thesame binding site on ryanodine receptor. J. Biol. Chem. 283, 26676 –26683

54. Xu, S., Bayat, H., Hou, X., and Jiang, B. (2006) Ribosomal S6 kinase-1modulates interleukin-1�-induced persistent activation of NF-�Bthrough phosphorylation of I�B�. Am. J. Physiol. Cell Physiol. 291,C1336 –1345

55. Tan, Y., Ruan, H., Demeter, M. R., and Comb, M. J. (1999) p90(RSK) blocksbad-mediated cell death via a protein kinase C-dependent pathway. J. Biol.Chem. 274, 34859 –34867

56. Shimamura, A., Ballif, B. A., Richards, S. A., and Blenis, J. (2000) Rsk1mediates a MEK-MAP kinase cell survival signal. Curr. Biol. 10, 127–135

57. Roux, P. P., Ballif, B. A., Anjum, R., Gygi, S. P., and Blenis, J. (2004) Tumor-promoting phorbol esters and activated Ras inactivate the tuberous scle-rosis tumor suppressor complex via p90 ribosomal S6 kinase. Proc. Natl.Acad. Sci. U.S.A. 101, 13489 –13494

58. Anjum, R., Roux, P. P., Ballif, B. A., Gygi, S. P., and Blenis, J. (2005) Thetumor suppressor DAP kinase is a target of RSK-mediated survival signal-ing. Curr. Biol. 15, 1762–1767

59. Cho, Y. Y., Yao, K., Bode, A. M., Bergen, H. R., 3rd, Madden, B. J., Oh, S. M.,Ermakova, S., Kang, B. S., Choi, H. S., Shim, J. H., and Dong, Z. (2007)RSK2 mediates muscle cell differentiation through regulation of NFAT3.J. Biol. Chem. 282, 8380 – 8392

60. Rosner, M., and Hengstschläger, M. (2008) Cytoplasmic and nuclear dis-tribution of the protein complexes mTORC1 and mTORC2: rapamycintriggers dephosphorylation and delocalization of the mTORC2 compo-nents Rictor and Sin1. Hum. Mol. Genet. 17, 2934 –2948

61. Sapkota, G. P., Cummings, L., Newell, F. S., Armstrong, C., Bain, J., Frodin,M., Grauert, M., Hoffmann, M., Schnapp, G., Steegmaier, M., Cohen, P.,and Alessi, D. R. (2007) BI-D1870 is a specific inhibitor of the p90 RSK(ribosomal S6 kinase) isoforms in vitro and in vivo. Biochem. J. 401, 29 –38

62. Vaidyanathan, H., and Ramos, J. W. (2003) RSK2 activity is regulated by itsinteraction with PEA-15. J. Biol. Chem. 278, 32367–32372

63. Heizmann, C. W. (1986) Intracellular calcium-binding proteins: structureand possible functions. J. Cardiovasc. Pharmacol. 8, S7–12

64. Heizmann, C. W. (2002) The multifunctional S100 protein family. Meth-ods Mol. Biol. 172, 69 – 80




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Stuart S. Martin, Paul T. Wilder and David J. WeberKira G. Hartman, Michele I. Vitolo, Adam D. Pierce, Jennifer M. Fox, Paul Shapiro,

Signal-regulated Kinase (ERK)-mediated Phosphorylation of RSK(RSK) in Malignant Melanoma Is Calcium-dependent and Inhibits Extracellular

Complex Formation between S100B Protein and the p90 Ribosomal S6 Kinase

doi: 10.1074/jbc.M114.561613 originally published online March 13, 20142014, 289:12886-12895.J. Biol. Chem.

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